ms2-affinity-purification-coupled-with-rna-sequencing-gram-positive

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

Université de Strasbourg, CNRS

RNA molecules with regulatory functions in pathogenic bacteria have benefited from a renewed interest these two last decades. In Staphylococcus aureus, recent genome-wide approaches have led to the discovery that almost 10-20% of genes code for RNAs with critical regulatory roles in adaptive processes. These RNAs include trans-acting RNAs, which mostly act through binding to target mRNAs, and cis-acting RNAs, which include regulatory regions of mRNAs responding to various metabolic signals. Besides recent analysis of S. aureus transcriptome has revealed an unprecedented existence of pervasive transcription generating a high number of weakly expressed antisense RNAs along the genome as well as numerous mRNAs with overlapped regions. Here, we will illustrate the diversity of trans-acting RNAs and illustrate how they are integrated into complex regulatory circuits, which link metabolism, stress response and virulence. 

The importance of regulatory RNAs in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive major human pathogen involved in a wide range of human infectious diseases (from minor skin infections to septicemia, endocarditis or toxic shock syndrome). The treatment of S. aureus infections is very challenging due to the emergence of multiple antibiotic-resistant isolates. The high diversity of clinical symptoms caused by S. aureus depends on the precise expression of numerous virulence factors and stress response pathways, which are tightly regulated at every level (transcriptional, posttranscriptional, translational, and posttranslational). During the last two decades, it has become evident that small regulatory RNAs (sRNAs) play a major role in fast adaptive responses, mainly by targeting mRNA translation. sRNAs act as antisense RNAs by forming noncontiguous pairings with their target mRNAs and their mechanisms of action vary according to the interaction site. To obtain a global and detailed view of the regulatory networks involved in the adaptive processes of S. aureus, we have adapted the MAPS approach to get individual sRNA targetomes. We also set up different strategies to validate MAPS results and establish sRNA regulatory activities. As this method has been first developed in Gram-negative bacteria, we provide here a protocol for its application in S. aureus and highlight underlying differences. Finally, we discuss several points that have been and could be further improved and provide a workflow file for the automatic analysis of the sequencing in Galaxy.

MS2-Affinity Purification Coupled With RNA Sequencing Approach in the Human Pathogen Staphylococcus aureus.

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of  mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.

Isabelle Caldelari

Université de Strasbourg, CNRS

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria